The goal of these studies is to elucidate factors that determine the spectral sensitivities of individual photoreceptor cells. In general, a photoreceptor's spectral sensitivity will depend upon the type of visual pigment expressed and the absorption properties of the pigment. Neither of these aspects is well understood. A visual pigment's spectral tuning is produced by perturbations of the chromophore by its specific opsin. The first aim of this proposal is to find out how and where opsin perturbs the chromophore to tune its absorption. My approach will be to characterize the spectral sensitivities of isolated rods and cones of salamander and goldfish electrophysiologically before and after replacing their native chromophore with chemically modified retinal analogs. Using a series of dihydroretinal analogs, we will localize the chromophoric sites that interact with opsin. The second aim is to find out whether a cell can express multiple opsins and whether all opsins form functional pigments. Although an individual photoreceptor is thought to express only one type of opsin, there is evidence to suggest that some photoreceptors violate this rule. We will test for the presence of two functional opsins in guinea pig cones and three in salamander cones by measuring the cell's spectral sensitivity before and after exposure to an intense bleaching light. The spectral composition of the bleaching light will be carefully chosen so that one pigment type is bleached preferentially. Additional evidence that multiple opsins are expressed will be obtained using immunohistochemical and molecular biological probes. The third aim is to investigate the ability of photoreceptors to change the type of opsin they express. We will measure the spectral sensitivities of cones in salamander and winter flounder at different ages to see if their pigment content changes as a function of time. The results of these studies could have developmental significance for the regulation of cone density and the formation of cone-specific neural circuits in the retina. The long term goal of these studies is to understand how the retina encodes visual information carried by wavelength and to explore the plasticity of the underlying mechanisms.